Method of in vitro fertilization with delay of embryo transfer and use of peripheral blood mononuclear cells

ABSTRACT

A method of in vitro fertilization wherein the embryo is implanted into the uterus of a female patient at least two, and preferably three to twelve months after the eggs are retrieved from the patient in order to reduce the effect of autoimmune rejection of the embryo by the patient&#39;s autoimmune system and increase the probability and success of pregnancy and wherein prior to embryo implantation, the endometrium in the uterus is prepared for embryo implantation by introducing peripheral blood mononuclear cells (PBMCs) into the uterus. The procedure is combined with cryopreservation techniques to preserve the oocytes or the IVF-produced embryos of the patient.

PRIOR APPLICATIONS

This application claims the benefit of prior-filed U.S. ProvisionalPatent Application Ser. No. 61/629,651, filed on Nov. 23, 2011, thesubject matter of which is hereby incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was not developed with the use of any FederalFunds, but was developed independently by the listed inventors.

FIELD OF THE INVENTION

The present invention relates to the field of in vitro fertilization,specifically in women who have reduced fertility as a result ofautoimmune reactions. Disclosed is a method used to enhance the rate andstability of pregnancy inception by combining in vitro fertilizationtechniques, optionally with prolonged cryopreservation of their oocytesor IVF-produced embryos, combined with controlled preparation of theendometrium in the uterus by means of peripheral blood mononuclear cells(PBMCs) prior to embryo transfer.

BACKGROUND OF THE INVENTION

The World Health Organization reports that each year, more than 15% ofwomen worldwide experience difficulties getting pregnant and seekmedical assistance (WHO 1997), estimated to be 60 to 80 million womenaround the world a decade ago. Infertility is generally defined by theWorld Health Organization as a lack of conception after an arbitraryperiod of twelve months. However, many couples attempt for years toconceive naturally before seeking medical assistance in an effort tobecome pregnant. A decrease in fertility rate is associated with medicaland non-medical factors. For example, women's age has been shown to be adirect major determinant of the average time required to conceive. Ithas been shown that premature ovarian failure occurs in 1:1000 womenbefore the age of 30; 1:250 women by 35 years; and 1:100 by the age of40. Therefore, the highest birth rates are in the age group of 25-30years and declines sharply after the age of 35 years. Infertility iscurrently one of the most frequent health concerns facing the populationaged 25-45 years. Thus, great interest and need exist for a method ofextending fertility in healthy women, possibly taking away age-relatedbarriers to childbearing, and for women who are unable to conceivethrough natural methods. Although infertility itself may not threatenphysical health, it often has a serious impact on the emotional, mentaland spiritual well-being of women and of couples.

Assisted reproductive technologies are procedures that involveextracorporeal handling of both human eggs (oocytes or ova) and sperm(spermatozoa), and of embryos for the purpose of establishing apregnancy in a female subject. These procedures include, but are notlimited to, in vitro fertilization (“IVF”) including embryo transfer,gamete intrafallopian transfer, zygote intrafallopian transfer, tubalembryo transfer, gamete and embryo cryopreservation, oocyte and embryodonation, and gestational surrogacy. In vitro fertilization (“IVF”) hasevolved as the major treatment for infertility or sub-fertility whenother methods of assisted reproductive technology have failed. In itsmost basic sense, the process involves extracting the female egg from awoman and fertilizing the egg by sperm outside the body (“in vitro”).The process involves monitoring a woman's ovulatory process, removingmultiple eggs from the woman's ovaries and letting sperm fertilize theeggs in a fluid medium in a laboratory. The eggs are usually retrievedfrom the patient by transvaginal oocyte retrieval involving anultrasound-guided needle piercing the vaginal wall to reach the ovaries.Through this needle, follicles can be aspirated, and the follicularfluid is handed to the IVF laboratory to identify and diagnose the ova.It is common to remove between ten and thirty eggs from each patient.The fertilized egg, (embryo), or usually multiple embryos, are thentransferred to the patient's uterus with the intention of establishing asuccessful pregnancy. See, for example, U.S. Pat. No. 7,781,207.

First developed in the 1970's, in vitro fertilization has provided aneffective form of assistance for a large proportion of women to date.Currently, it is reported that IVF accounts for 1.3% of all live birthsin Europe [Nygren et al. 2001] and 1.7% of all live births inAustralasia [Hurst et al. 2001.] In the United States, assistedreproductive technology IVF cycles started in 2006 resulted in 41,343births (54,656 infants), which was slightly more than 1.0% of total USbirths that year. In 2010, Robert Edwards was awarded the Nobel Prize inPhysiology or Medicine for the development of in vitro fertilization.The next step was the ability to freeze and subsequently thaw andtransfer embryos, first pioneered by Carl Wood, which significantlyimproved the feasibility of IVF treatment.

Typical successful pregnancy rates obtained with previous in vitrofertilization techniques remain relatively low, in the range of 15% to25%, based on a number of reported studies. It is widely accepted byhealth care professionals that the most significant limiting factor tofertilization is the failure of embryos to implant into the endometrium,the lining of the uterus. Blake et al. report that 80-85% of embryosfail to result in pregnancy following transfer into IVF patientsresulting in an enormous wastage of embryos. Simon et al. demonstratedthat in women undergoing IVF cycle, implantation was detected in 60% ofthe cycles, therefore in all embryos transferred during IVF, 40% fail toimplant.

One possible reason for low implantation rates during IVF is thatembryos are transferred to the uterus two days after fertilization atthe 4-8 cell stage. One view is that it may be more desirable to useembryos at the blastocyst stage reached at day 5-7 of culture. Theadvantages suggested include improved synchronization between embryo anduterus and the ability to select better quality embryos over the longerculture period. Blastocyst transfer may also help reduce the number ofmultiple births resulting from IVF by allowing the selection of fewernumbers of highly competent embryos per transfer. Typically during IVFprocedure, two to five embryos are transferred to the uterus in order toincrease the chance of implantation, creating the risk of multiplepregnancies. Consequently, more than half of all babies born in theUnited States after IVF treatment result from multiple gestations. It issometimes the case that multiple implantations occur in the uterus andas the embryos continue to grow and develop within the womb, the patientis unable to carry more than one or two fetuses to term. At that point,a difficult decision is made to terminate those pregnancies and withdrawthe additional fetus or fetuses from the womb, a process called embryoor fetus reduction, a procedure to reduce the number of viable embryosor fetuses in a multiple pregnancy. Such decisions carry with them bothethical and often psychologically traumatic implications. Development oflaboratory techniques which would increase the probability and certaintyof implantation of each embryo are still desired for a number of theaforementioned reasons.

In a process called natural cycle in vitro fertilization, thefertilization is performed by collecting one or more naturally selectedeggs from the patient during a woman's natural menstrual cycle withoutthe use of any drugs. In modified natural cycle IVF, fertilitymedication is used for two to four days during a woman's natural cycleto avoid spontaneous ovulation and to make the treatment moresuccessful. “Mild IVF” is a method where a small dose of ovarianstimulating drugs are used for a short duration during a woman's naturalcycle aimed at producing 2-7 eggs and creating healthy embryos. Thismethod appears to reduce complications and side-effects for women and itis aimed at quality, and not quantity of eggs and embryos. However, thismethod yields a very low success rate of pregnancy.

Additional techniques are therefore used routinely in order to increasethe chance of pregnancy. The most common is ovarian hyperstimulation orsuper-ovulation that is used in order to stimulate the ovaries toproduce multiple eggs that are then retrieved from the patient. The longprotocol typically involves downregulation (suppression or exhaustion)of the pituitary ovarian axis by the prolonged use of a gonadotropinreleasing hormone (GnRH) agonist. Subsequent ovarian hyperstimulation,typically using follicle stimulating hormone (FSH), starts once theprocess of downregulation is complete, generally after 10 to 14 days. AnIVF cycle using this protocol is known as conventional in vitrofertilization. The short protocol skips the downregulation procedure,and consists of a regimen of fertility medications to stimulate thedevelopment of multiple follicles of the ovaries. Other procedures usegonadotrophin-releasing hormone agonists (GnRHA), which decreases theneed for monitoring by preventing premature ovulation, and more recentlygonadotrophin-releasing hormone antagonists (GnRH Ant) have been used,which have a similar function. In most patients, injectablegonadotropins (usually FSH analogues) are used under close monitoring.Such monitoring frequently checks the estradiol level of the patientand, by means of gynecologic ultrasonography, follicular growth.Typically, approximately 10 days of injections are necessary. Ovarianstimulation carries the risk of excessive stimulation leading to ovarianhyperstimulation syndrome (OHSS), a potentially life-threateningcomplication of abdominal distension, ovarian enlargement, andrespiratory, hemodynmic and metabolic complications. In addition, it hasalso of recent been demonstrated that fertility drugs used for thestimulation of ovulation of patients undergoing IVF treatment contributeto compromised implantation receptivity of the embryo in the uterus andlead to a decreased rate of pregnancy inception. [Ertzeid et al. 2001].

Female fertility can be affected by dysfunctions of the reproductivetract, of the neuroendocrine system or of the immune system. Some femalecancer patients risk losing their fertility because certain kinds ofchemotherapy treatments, and certain types of radiation treatments, canbring on premature menopause rendering them sterile. In Western Europeand North America, endocrine dysfunction is identified in about 10 to20% of women presenting with infertility [Crosignani et al. 2000] Still,in about 10 to 20% of cases, the cause of infertility remains unknown.It is postulated that autoimmune reactions of the body may be the causeof infertility in many such women. Reproductive autoimmune failure anddefects can be associated with overall activation of the immune systemor with immune system reactions that are specifically directed againstovarian antigens.

Haller-Kikkatalo explains that active tolerance mechanisms are requiredto prevent self-protective inflammatory responses in the human body tothe many foreign air-borne and food antigens that are encountered at thebody's mucosal surfaces. However, the most important aspect of toleranceis self-tolerance, which prevents the body from mounting and immuneattack against its own tissues—this is the prevention from autoimmunereactions. Autoimmunity is associated with an imbalance of variouscomponents of immune response and with the development of autoantibodiesdirected against normal host antigens. Female fertility is regulated bya series of highly coordinated and synchronized interactions in thehypothalamic-pituitary-ovarian axis. Reproductive autoimmune failuresyndrome was originally described by Gleicher et. al. in women withendometriosis, infertility and increased autoantibodies. Although theimpact of particular autoantibodies on the pathogenesis of infertilityis not yet uniformly understood, autoimmune mechanisms as well as anincreased production of multiple autoantibodies are involved in suchinfertility disorders as premature ovarian failure (POF), subclinicalovarian failure, recurrent pregnancy loss, endometriosis, polycysticovary syndrome (PCOS), unexplained infertility, repeatedly unsuccessfulIVF attempts, and spontaneous abortions. Some studies have suggested thelesser importance of specific antibodies and stressed the key role ofoverall activation of the immune system in reduced fecundity. [N.Gleicher, 2001; Dmowski et al., 1995.]

A group of research has focused on developing approaches to overcomeimmune-related infertility. The first and most commonly used approach isthe use of medications that aim to suppress the autoimmune response inthe patient in order to allow inception of pregnancy. For example, lowdose oral prednisolone therapy was suggested for improving pregnancyrate in patients with recurrent IVF failure. However, contradicting dataexist indicating that certain antibodies damage the embryo, interferewith the implantation process or interfere with the formation of theplacenta. This renders it difficult to predict the success of therapyusing these particular immunosuppressive medications. Methods havedeveloped using milder pretreatment with acetylsalicylic acid orheparin. However, though this treatment has become generally universal,the rate of pregnancy using this approach remains relatively low.[Maghraby et al., 2007]

A second approach has recently been introduced. The procedure involvesthe preparation both of the endometrium and its surrounding environmentby means of peripheral blood mononuclear cells (PBMCs). [Fujiwara etal., Kosaka et al., Yoshioka et al.] Peripheral blood mononuclear cells(PBMCs) allow the endometrium of the uterus to grow in sufficient sizeprior to embryo implantation and also act as building blocks by the bodyfor growing the embryo(s) after their implantation in the uterus of thewoman. According to proponents, PBMCs have been identified asmultipotent cells. Multipotent cells produce cells of a particularlineage or closely related family. They have been shown to have thecapability to be naturally transformed into any kind of human tissue.Multipotent cells are a valuable resource for research and therapeutictreatments. Recent advances in bioengineering are quite promising inrepairing, building, engineering, regenerating, generating and growingtissue.

European Pat. Application EP1581637 discloses monocyte derived adultstem cells that are isolated from peripheral blood of mammals andmethods of preparing, propagating and using these stem cells. Theinventors of U.S. Pat. No. 7,795,018, M. Kuwana and H. Kodamo, disclosemonocyte-derived multipotent cells (MOMCs) that can differentiate intoendothelial cells by a medium culture under conditions inducingdifferentiation into endothelium. Further disclosed is a method forpreparing MOMCs involving culturing PBMCs in vitro on fibronectin andcollecting fibroblast-like cells. It was demonstrated that by culturingin a EBM-2 medium, a maintenance medium of endothelial cells, for sevendays, MOMCs differentiate into endothelial cells changing the cell'smorphology from a spindle shape to a morphology having multipleprojections (see U.S. Pat. No. 7,795,018). The researchers of thepresent invention hypothesized that the use of PBMCs can therefore beinstrumental during the reproductive process and have incorporated theuse of PBMCs into a novel technique of in vitro fertilization providedherein.

A group of various inception agents has been developed, which separatelyor in combination, can be applied during IVF treatment in order topromote the acceptance of the embryo by a woman's immune system. Amongthese agents, soluble human leukocyte antigen G (sHLA-G) appears to bepromising. The s-HLA class of molecules has been recognized to beinvolved in immune response and in the modulation of the maternal-fetalimmune relationship during pregnancy. Sher et al. in U.S. patentapplication Ser. No. 10/829,081 discloses having isolated sHLA-G fromthe culture media surrounding pooled developing embryos and blastocysts.They observed that the absence of sHLA-G in the supernatant surroundinggroups of embryos in culture is associated with significantly reducedIVF implantation and pregnancy rates. They proposed that addition ofsHLA-G to the medium in which embryos are cultured and/or delivered intothe uterine environment through embryo transfer, would enhanceimplantation and pregnancy potential of those embryos. Though thetechnique is useful, neither this approach, nor any other alone hassolved the problem of autoimmune infertility. It is suggested hereinthat such inception agents be used in conjunction with the procedure ofthe invention herein to improve the probability and success of pregnancyinception.

Finally, engineered glycolipid-like molecular constructs have been shownto be capable of modifying the embryos and enhancing the interactionbetween the embryo and the target tissue, the endometrium. Building of amacro-molecular matrix on the external surface of an embryo and loadingof said matrix with certain agents for stimulation of pregnancyinception are the techniques that, according to proponents, will havevery broad applications in IVF treatment in the future. Not only has themodification of embryos by this constructive approach been successfullydemonstrated in an in-vitro culture system, but animals have given birthto healthy offspring derived from such modified embryos. However, thisapproach has not yet been tested in clinical trials and remainspremature to be considered among current medical tools.

Much research has been directed to procedures for improving theprobability of successful pregnancy and birth of a child. Despite theconsiderable research, technical advances and variations in procedures,the rate of successful pregnancy utilizing IVF treatment still remainson average in the order of 15-25% per cycle. The researchers herein haveattempted to address the challenge of embryo implantation and decreasethe risk of autoimmune system responses by presenting a solutionfocusing on stabilizing the interaction between a woman's immune systemand the embryo.

SUMMARY OF THE INVENTION

In its broadest sense of the invention, provided is a method of in vitrofertilization for a female patient, the method comprising the steps ofintroducing into the uterus an effective amount of a compositioncomprising peripheral blood mononuclear cells, and transferring at leastone embryo into the uterus of the patient after a predetermined delayfollowing initiation of the in vitro fertilization of said patient; andwherein the method results in an increase in the probability ofimplantation of the embryo in the uterus with successful inception ofpregnancy when compared to in vitro fertilization methods lacking suchsteps. The predetermined delay of time is a period of time sufficient todecrease autoimmune rejection of the embryo or the risk of autoimmuneresponse of the patient. The preferred delay of time before embryotransfer is at least two menstrual cycles or two cycles of ovulation ofthe patient, though the delay will vary from patient to patient andamong species, hybrids and varieties of animals. The preferred delay oftime in a human patient is three to twelve months.

It is another aspect of the invention that toward the end of thetime-delay period, the women's endometrium is prepared in a way so as tooptimize the acceptance of the embryo by the uterine cavity. Accordingto the invention, this is accomplished by intrauterine injection ofperipheral blood mononuclear cells (PBMCs), most preferably obtainedfrom the patient. It has been observed herein that this method increasesthe probability of successful pregnancy inception and early pregnancydevelopment. Combining both the time-delay of implantation inconjunction with cryopreservation of the embryo and the PBMCspreparation of the woman's endometrium is the foundation of the presentinvention.

More particularly, disclosed is a method of in vitro fertilization for afemale patient involving the steps of: (a) obtaining at least one oocyteand fertilizing the oocyte with spermatozoa to form a zygote; (b)developing the zygote in vitro to an embryo stage; ((c) cryopreservingthe embryo; (d) waiting a predetermined period of time sufficient todecrease the risk of autoimmune response of the patient; (e) extractinga first portion of peripheral blood mononuclear cells (PBMCs) from theblood of the patient 2 to 4 days prior end of waiting period; (f)culturing said first portion of PBMCs in a suitable culture medium c(g)extracting a second fresh portion of PBMCs from the blood of the patienton the last day of the waiting period; (h) combining the cultured firstportion of PBMCs with the fresh second portion of PBMCs to obtain acomposition comprising fresh and cultured PBMCs; (i) introducing thecomposition of PBMCs into the uterus of the patient; (j) thawing theembryo from the cryopreserved state; and (k) transferring at least onethawed embryo into the uterus of the patient to effectuate pregnancy.

An additional aspect of the invention is a composition comprising PBMCs,a method for producing the composition and the application of the PBMCcomposition in IVF treatment and for growing and engineering certaintarget tissue of an organism, most preferably the endometrium of afemale uterus. Such method involves extracting PBMCs from the blood of apatient; propagating a portion of the extracted PBMCs in the presence of4.8-6.0% carbon dioxide (CO2) at 36.7-37.3° C. in a culture mediumcontaining (i) RPMI 1640 medium with L-glutamine and sodium bicarbonate,(ii) human recombinant albumin and (iii) a promoting agent capable ofimproving the ability of PBMCs to enhance tissue growth, such as humanchorionic gonadotropin (hCG); and combining the fresh and culturedportions of PBMCs to obtain said composition.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be more clearly understood by definingcertain terms that are used and relied upon throughout thespecification.

“Blastocyst” is an embryo, five or six days after fertilization, havingan inner cell mass, an outer cell layer called the trophectoderm, and afluid-filled blastocele cavity containing the inner cell mass from whichthe whole of the embryo is derived. The trophectoderm is the precursorto the placenta. The blastocyst is surrounded by the zona pellucidawhich is subsequently shed when the blastocyst “hatches.” The zonapellucida, composed of a glycoprotein coat, surrounds the oocyte fromthe one-cell stage to the blastocyst stage of development. Prior toembryo attachment and implantation, the zona pellucida is shed from theembryo by a number of mechanisms including proteolytic degradation. Thezona pellucida functions initially to prevent entry into the oocyte bymore than one sperm, then later to prevent premature adhesion of theembryo before its arrival into the uterus.

“Cryo-IVF” refers to a process of in vitro fertilization in which theembryo is cryopreserved then thawed prior to embryo transfer or aprocess in which the oocyte used for fertilization has been previouslyfrozen then thawed. “Fresh-IVF” refers to a process of in vitrofertilization wherein the embryo is not frozen prior to transfer intothe uterine cavity and where the oocytes used to prepare the embryo havenot previously been frozen.

“Embryo” is the product of the division of the zygote to the end of theembryonic stage, eight (8) weeks after fertilization. The cleavage stageof the embryo occurs during the first three days of culture. “Embryotransfer” is the procedure in which one or more embryos and/orblastocysts are placed into the uterus or fallopian tubes. As such, theterms “blastocyst” and “embryo” are used interchangeably herein forpurposes of defining the term “embryo transfer” and in application ofthe term “embryo transfer” within the scope and application of theinvention as described and claimed.

“Endometrium” refers to the tissue lining the internal surface of theuterus, which is composed of a layer of epithelial cells. The embryocomes into contact with the endometrium and the extracellular matrix(the “mucus”) for implantation. The epithelial and underlying stromalcell layer cyclically thickens, secretes mucus and is shed from the bodyunder the hormonal influences of the menstrual cycle. By the term“implantation” herein is meant the attachment and subsequent penetrationby the blastocyst (after having shed its zona pellucida), usually intothe endometrium. Attachment to the endometrium lining may occur byinteraction between the attachment molecules and on or more componentsof the endometrium, including membranes of the epithelial cells, mucus,mucin components of the mucus, or an exogenously introduced component ofthe uterus.

“Fertilization” refers to the penetration of the ovum by the spermatozoaand combination of their genetic material resulting in the formation ofa zygote. “Initiation of in vitro fertilization” as used herein meansthe initiation of controlled ovarian stimulation of a female patient,which involves pharmacological treatment in which women are stimulatedto induce the development of multiple ovarian follicles to obtainmultiple oocytes at follicular aspiration.

“Uterus”, commonly referred to as the womb, is the major femalehormone-responsive reproductive sex organ of most mammals includinghumans that contains the cervix at one end while the other is connectedto one or both fallopian tubes, depending on the species. Thereproductive function of the uterus is to accept a fertilized ovum whichpasses through the utero-tubal junction from the fallopian tube. Itimplants into the endometrium, and derives nourishment from bloodvessels which develop exclusively for this purpose. The fertilized ovumbecomes an embryo, attaches to a wall of the uterus, creates a placenta,and develops into a fetus during gestation until childbirth. As usedherein, the term “uterus” incorporates the fallopian tubes for purposesof embryo transfer. The term “uterus” is also used interchangeablyherein with the term “uterine cavity,” which is the cavity of the bodyof the uterus.

During IVF treatment, the immune system of a woman experiences astimulated production and delivery of oocytes. If the IVF-producedembryo is transferred into the uterus cavity shortly after extraction ofoocytes, the immune system may generate an overreaction. Suchoverreaction of the immune system can be the cause of the body'sinability to implant the embryo and lead to autoimmune infertility. Inits first aspect, the invention presents delaying the introduction ofthe embryo into the uterus in order to allow the woman's immune systemto settle and regain its proper hormonal balance. During the time-delay,the female patient's oocytes or embryos are optionally preserved viacryopreservation.

The time-delay useful in the method of the invention herein is apredetermined amount of time for each specific patient. For humanpatients, the time delay may be a length of time anywhere of at leasttwo months or two cycles of ovulation. In the preferred embodiment, thepredetermined delay is three cycles of ovulation, which typicallycorresponds to three months. Most women experience one cycle ofovulation every 28 days. It is to be understood, however, that becausecycles of ovulation vary from woman to woman, the number of actual daysof the delay will vary with each patient in the course of treatment ofthe IVF procedure of the invention. It is possible that a female patientwill experience only two cycles of ovulation within a three-monthperiod, or likewise, that a woman may experience more than three cyclesof ovulation within a three-month period. In some cases, it is possiblethat a patient will not experience any additional ovulation after theinitial ovulation post initiation of the IVF treatment. The time delaycan also be of duration of more than three months, but will preferablybe within three months to one year. It has been demonstrated herein thatthe predetermined time-delay significantly decreases the risk ofautoimmune infertility and, therefore, significantly increases theprobability of successful pregnancy

Within the scope of the invention are embodiments wherein oocytes orembryos that are derived from a female or females other than the patientare used for pregnancy inception, termed “donor eggs” or “donorembryos”, respectively. This would occur in situations where the femalepatient is not able to ovulate or to produce viable ova, or in which theembryos derived by fertilizing the patient's own eggs are determined tobe too deficient for embryo transfer or have a low probability ofsurvival based on either morphological or genetic testing.

In its common technique, women undergoing IVF require hormone injectionsto stimulate follicular development and multiple egg production. Thisstimulation process usually requires the initial use of a gonadotropinreleasing hormone (GnRH) agonist to suppress ovarian function,preventing ovulation until the desired time. The medication which ismost commonly used is clomiphene citrate CLOMID®, (Aventisub, LLC,Greenville, Del., US), a selective estrogen receptor modulator thatincreases production of gonadotropins by inhibiting negative feedbackfrom estrogen on the hypothalamus. This induction of final maturationand release of oocytes is often induced by administration of luteinizinghormone. Protocols for these injections are well known and establishedin the art and are utilized in any of the embodiments of the methods ofthe invention.

According to the method of the invention, unfertilized eggs areharvested and retrieved from the female patient by techniques known inthe art. Such techniques involve placing a specially-designed needleinto the ovarian follicle and removing the fluid that contains the eggs.Once the follicular fluid is removed from the follicle, the eggs may beinspected microscopically and diagnosed to observe their morphologicalfeatures. One method for obtaining oocytes for IVF is disclosed in U.S.Pat. No. 4,725,579. In the method herein, six oocytes are gathered fromthe patient, though preferably four are obtained. The eggs are thenplaced into an incubator. Conventional insemination or intracytoplasmicsperm injection (ICSI) is used to fertilize the eggs. The type offertilization used is often based on the male's semen parameters orother factors such as the type of analysis that may be required of theembryo. During conventional insemination, sperm are mixed with the eggsin a culture dish and incubated overnight to undergo the fertilizationprocess. During intracytoplasmic sperm injection, one sperm is directlyinjected into one egg. With either technique, the eggs are observed thefollowing day to evaluate for cell division. The fertilized eggs, nowcalled embryos, are then placed into specific culture media that promotegrowth and development.

According to the invention, the IVF-derived oocytes or embryos are grownon suitable culture medium. Available culture media attempt to providethe nutrients needed for cells to grow and develop and seek to duplicateas much as possible the conditions normally occurring within the femalereproductive system. Culture media known in the art that are suitablefor use for the in vitro support of cell development and growth inlaboratory procedures can be used herein. Examples include, but are notlimited to human tubal fluid (HTF) (Irvine Scientific),N-2-hydroxyethylpiperazine-N′-2-ethane (HEPES) media (IrvineScientific), IVF-50 (Scandanavian IVF Science), S2 (Scandanavian IVFScience), G1 and G2 (Scandanavian IVF Science), UniIVF, ISM-1,BlastAssist, UTM media (sold as MEDICULT® media by Origio A/S), ModifiedWhittens medium, Wittinghams T6 media, Ham's F-10 media, Earle'ssolution. Buffering systems, such as 4-morpholinepropanesulfonic acid(MOPS) are typically provided. The procedures are well set forth inTrouson et al. (1980 and 1982) and Quinn et al. (1985).

Tissue culture media generally are complicated systems, containing anarray of amino acids, vitamins and other constituents. Some mediaconsist of balanced salt solutions supplemented with carbohydrate energysources such as glucose, pyruvate and lactate. The media can also besupplemented with non-essential amino acids. Components in the mediumare often derived from non-human and non-animal sources such asrecombinant microorganisms. It is desirable to take steps to reduce thepossibility of contamination, such as proper purification andpreparation techniques practiced in the art. In addition, media mayinclude antibiotics, such as penicillin or streptomycin to destroybacteria that might be introduced into the medium during the process ofoocyte collection.

During in vivo development within the female reproductive system, theoocyte is originated within and released from the ovary during ovulationand proceeds through the oviduct toward the uterus. The fluid of theoviduct contains a number of components that provide nourishment to theoocyte and its surrounding cumulus cells. Once fertilization occurs, theresulting zygote travels down the oviduct and enters the uterusapproximately three days later, undergoing internal transformation andexperiencing a changing environment. Significant development changesoccur as the zygote/embryo/blastocyst develops. The composition of thefluid surrounding the developing embryo in the uterus is tailored tothose changing needs.

No single medium is optimized for supporting the gametes, fertilization,zygote maturation, and embryo development compared to the naturalenvironment of the female reproductive system. Thus a number ofspecialized media have become available to address the varying stages ofembryo development. For example, G1 and G2 media were specificallyformulated to meet the physiological needs of the cleavage stage embryoand the embryo in the eight-cell through blastocyst stage ofdevelopment. U.S. Pat. No. 6,605,468 to Robertson et al. discloses amedium for the propagation of early stage embryos to blastocyst stage.The medium contains an effective amount of granulocyte-macrophagecolony-stimulating factor (GM-C SF) to increase the percentage ofpre-blastocyst embryos which develop to transfer-ready blastocysts. Alsodisclosed is a method of growing early stage human embryos to transferready blastocysts. The result is that a greater proportion of embryoscan be grown to blastocyst stage and be used for implantation in an IVFprogram. A number of studies have been made using culture techniqueswhereby the embryos are co-cultured with feeder cells. [Menezo et al.,1990; Planchot et al., 1995] Other stimulatory factors such as cytokinescan be added to the medium to propagate embryo growth, such as leukemiainhibitory factor (LIF), see for example U.S. Pat. No. 5,418,159 toGough et al. Leukemia inhibitory factor is a potent hormone havinggeneral utility in the area of in vitro embryology, such as inmaintaining embryonic stem cell lines and increasing the efficiency ofembryo transfer.

In U.S. Pat. No. 6,838,235, Gardner et al., provide that instead ofimmersing human reproductive cells in a single culture medium throughoutthe IVF process, the reproductive cells may be moved through a sequenceof distinct culture media as the various IVF procedures are carried out.In one formulation, the culture media is specifically formulated toprovide a physical environment similar to that found within the femalereproductive tract and conducive to growth and development of embryos.The disclosure suggests that specialized media can be provided foroocyte retrieval and handling; oocyte maturation; ordinaryfertilization; oocyte, zygote and embryo examination and biopsy;embryonic development to the eight-cell stage; embryonic development tothe blastocyst stage; embryo transfer; and cryopreservation.

In addition to the use of commonly available culture media for embryopreparation, the method of the invention can be combined with othertechniques in order to increase the probability and success of pregnancyinception. A number of methods have developed in which the embryo ismodified in some way prior to transfer into the uterine cavity. Forexample, European Pat. App. EP 1765987 (WO 2005121322 A1) disclosesenzymatic modification of cell-surface H antigen, by the addition of oneor more monosaccharide units generating cells which are serologicallyequivalent to A or B antigen red blood cells. Research indicates thatthere is a receptor on the embryo for hyaluronate and that there is alsoa receptor for hyaluronate on the endometrium of the uterus of themother. Hyaluronate is thought to act like biological glue that assiststhe embryo in binding to the endometrium and, accordingly, supportsimplantation. U.S. Pat. No. 8,183,214 to Carter et al. discloses amethod of localizing hyaluronic acid to the surface of a cell ormulti-cellular structure (an embryo) for use in IVF procedures. Thedisclosure provides carbohydrate-lipid constructs that incorporatestably into the lipid bi-layer or membrane of a cell or embryo changingits biological activity in order to improve certain characteristics,such as growth characteristics, storage characteristics, and survival ofthe embryo and the likelihood of implementation of the embryo followingtransfer to the uterus. Similarly, U.S. Pat. No. 7,819,796 to Blake et.al. discloses another exogenously prepared construct to enhance theattachment and implantation of an embryo. The embryo is modified byglycolipids having lipid tails that are inserted in to the cell membraneof the embryo or into the zona pellucida in which the glycolipid hasbeen modified to incorporate a binding part wherein the binding part isadapted to enable binding to an attachment molecule. Attachment of theembryo to the endometrium can occur through the binding parts directlyor through a bridging molecule. Another method, protein painting, is amethod for modifying the external antigens of cell membranes withoutgene transfer. The specification for international applicationPCT/US98/15124 (publication WO 99/05255) describes the enhancement ofimplementation by contacting the embryo with a lipid-modified adhesionmolecule so as to modify the development of the embryo. U.S. patentapplication Ser. No. 13/067,021 describes another non-transgenic methodfor effective qualitative and/or quantitative changes in the surfaceantigens expressed by a cell. The synthetic molecule constructsdisclosed by the researchers incorporate into the lipid bilayer of thecell, and it is proposed that such insertion is thermodynamicallyfavored. The foregoing methods of embryo preparation, as well as othermethods practiced in the art, are contemplated within the scope of theinvention herein.

In the traditional IVF process, embryos are transferred to the uterinecavity two days after fertilization when each embryo is at the four (4)cell stage or three days after fertilization when the embryo is at theeight (8) cell stage. It has been recognized that it may be desirable touse embryos at the blastocyst stage when reached at day five to seven ofculture. The IVF method of the invention herein allows for embryotransfer at any time along the spectrum of embryo/blastocystdevelopment. Through visual observation, such as by with the use ofmicroscopy, blastocysts or embryos are considered ready to betransferred to the uterus when the blastoceol cavity is clearly evidentand comprises greater than 50% of the volume of the embryo. In an invivo environment, this stage would normally be achieved four to fivedays after fertilization, soon after the embryo has traversed thefallopian tube and arrives in the uterus.

According to the second aspect of the invention, it has been discoveredby the inventors herein that introducing a composition containingperipheral blood mononuclear cells (PBMCs) into the uterus of a femalepatient prior to embryo transfer during IVF treatment clearly increasesthe probability of successful embryo implantation in the endometrium ofthe uterus thus leading to viable pregnancy. Without intending to bebound by scientific theory, it is thought that the PBMCs promote thehealth and viability of a woman's endometrium and of the transferredembryo.

PBMCs are multipotent progenitor cells that have the potential to giverise to cells from multiple, but a limited number of lineages. At theend of the long series of cell divisions that form the embryo are cellsthat are terminally differentiated, or that are considered to bepermanently committed to a specific function. Stem cell experiments havebeen able to direct blood stem cells to behave like neurons, or braincells—a process known as trans-differentiation. The use of pluripotentstem cells from fetuses, umbilical cords or embryonic tissues derivedfrom in vitro fertilized eggs raises ethical and legal questions in thecase of human materials, poses a risk of transmitting infections and/ormay be ineffective because they may be rejected by a recipient's immunesystem. Arguably, the use of PBMCs raises less ethical and legalconcerns because PBMCs are cells that are obtained from the blood and donot constitute stem cells, while still being able to differentiate.

As used herein, a peripheral blood mononuclear cell (PBMC) is amultipotent cell that is extracted, harvested, derived, isolated orotherwise obtained from the blood of a subject. A PBMC is a blood celland that has a round nucleus. The PBMC class includes, but is notlimited to lymphocytes, a monocytes and macrophages. These blood cellsare a critical component in the immune systems of organisms utilized infighting infection and in operating other functions involving the immunesystem. The lymphocyte population consists of T cells (CD4 and CD8positive ˜75%), B cells and NK cells (˜25% combined). The PBMCpopulation also includes basophils and dendritic cells.

PBMCs can be isolated from human peripheral blood by common methodsknown in the art. The cells can be extracted from whole blood usingFICOLL® (GE Healthcare Bio-Sciences AB LLC of Sweden), a hydrophilicpolysaccharide that separates layers of blood, which will separate theblood into a top layer of plasma, followed by a layer of PBMCs and abottom fraction of leukocytes, erythrocytes, and polymorphonuclear cells(such as neutrophils, eosinophils). The polymorphonuclear cells can befurther isolated by lysing the red blood cells. FICOLL® is part ofFICOLL-PAQUE® (GE Healthcare Bio-Sciences AB LLC of Sweden).FICOLL-PAQUE® is normally placed at the bottom of a conical tube, andblood is then slowly layered above the FICOLL-PAQUE®. After beingcentrifuged, several layers will be visible in the conical tube, fromtop to bottom: plasma and other constituents, the layer of mononuclearcells containing the PBMCs, FICOLL-PAQUE®, and erythrocytes andgranulocytes which are present in pellet form. This separation allowseasy harvest of the PBMC's. Some red blood cell trapping (presence oferythrocytes and granulocytes) may occur in the PBMC or FICOLL-PAQUE®layer. Major blood clotting may sometimes occur in the PBMC layer.Ethylene diamine tetra-acetate (EDTA)) and heparin are commonly used inconjunction with FICOLL-PAQUE® to prevent clotting. BecauseFICOLL-PAQUE® layering is a very slow process, devices that aid in theoverly, which is most time-consuming step, have been developed. One sucha product is SEPMATE™−50 (StemCell Technology Inc. of Canada), aspecialized tube containing a porous insert that forms a physicalbarrier between the FICOLL-PAQUE® and blood sample. This allows theblood sample to be rapidly pipetted onto the insert, avoiding the needfor overlaying it directly onto FICOLL-PAQUE®. The SEPMATE™ (StemCellTechnology Inc. of Canada) insert also reduces the duration of thecentrifugation step, and after centrifugation, the top layer containingplasma and PBMCs can be poured into a separate container. Other devicesinclude a column containing a porous high-density polyethylene barrieror “frit.” These products allow blood to be layered on much more quicklywithout mixing polysaccharide and blood. An example of such a product isthe “Accuspin System Histopaque-1077” sold by Sigma Aldrich. It is alsopossible to have the FICOLL-PAQUE® separating system included in avacutainer blood collection tube. Such vacutainers increase theconvenience and safety of collecting blood products, but are much morecostly than the basic vacutainer. Another such product, FLOATIES™, hasbeen shown to effectively overlay blood or a cellular suspension onFICOLL® using a special mixture of polymer beads or pellets. Thisproduct is inexpensive, reduces researcher reliance on technique, andactually speeds up the overlay process. Any of the foregoing techniques,including other techniques that are or will become practiced in art ofcollecting, isolating, extracting, harvesting, separating, removing, orin any other way obtaining PBMCs from the blood of an organism, human oranimal, are contemplated within the scope of the embodiments of theinvention herein.

In one of the aspects of the invention, provided herein is a method ofculturing the PBMCs cells. The method comprises culturing the cells onfibronectin-coated plates in a humidified atmosphere containing from4.8% to 6.0% of carbon dioxide (CO2) at a temperature in the range of36.7° C. to 37.3° C., at a density of 104 to 107/mL. In a preferredembodiment of the method, PBMCs are cultured for a period of time in therange of 46 hours to 72 hours. In a most preferred embodiment, the PBMCsare cultured on fibronectin-coated plates in an atmosphere of 5.0% ofCO2 at a temperature of 37.1° C. for 48 hours.

The culture media used herein for propagating the extracted PBMCs is aRoswell Park Memorial Institute medium, commonly known as RPMI medium,available from a number of sources. RPMI medium is often used for celland tissue culture. RPMI 1640 medium has traditionally been used for thegrowth of serum-free human lymphoid cells, bone marrow cells andhybridoma cells. RPMI 1640 medium uses a bicarbonate buffering systemand differs from most mammalian cell culture media in its pH 8formulation. The preferred medium according to the methods of theinvention utilizes RPMI 1640 culture medium containing L-glutamine andsodium bicarbonate.

According to another aspect of the invention, human recombinant albumin(HRA) is added to the culture medium. HRA is a well-known carrierprotein present in high concentrations in plasma with a circulatoryhalf-life of approximately 19 days. It functions in fatty acidtransportations to tissues, protein stabilization, binding metal ions tosurfaces, and an antioxidative effect in plasma. HRA is widely availablefrom a number of providers, for example from Novozymes, Inc. orSigma-Aldrich, LLC. The addition of HRA during the method of theinvention to the culture medium acts as a food supplement that improvesthe growth of the PBMCs.

In a further aspect of the invention, human chorionic gonadotropin (hCG)added to the RPMI 1640 culture medium herein. Human chorionicgonadotropin (hCG) is released in the body into maternal circulationduring pregnancy by placental synctiotrophoblasts. It has been shown tobe an immuno-modulating hormone and reported that administration of hCGincreases pregnancy success and antisera to hCG inhibits fetalimplantation. hCG interacts with certain receptors (LHCG receptors) andpromotes the maintenance of the corpus luteum during the beginning ofpregnancy, causing it to secrete the hormone progesterone. Progesteroneenriches the uterus with a thick lining of blood vessels and capillariesso that it can sustain the growing embryo and fetus. Due to its highlynegative charge, hCG may repel the immune cells of the mother,protecting the fetus during the first trimester. It has also beenhypothesized that hCG may be a placental link for the development oflocal maternal immunotolerance. For example, hCG-treated endometrialcells induce an increase in T cell apoptosis (dissolution of T cells).These results suggest that hCG may be a link in the development ofperitrophoblastic immune tolerance, and may facilitate the trophoblastinvasion, which is known to expedite fetal development in theendometrium. [Kayisli et al., 2003]. It has also been suggested that hCGlevels are linked to the severity of morning sickness in pregnant women.The minimum concentration of the hCG in the culture medium of theinvention is not less than 5 IU/mL For the purpose of clarity, it hasbeen found herein that the hCG in the culture medium prepared accordingto the method of the invention functions as a promoting agent thatimproves the ability of PBMCs to enhance tissue growth, specifically thegrowth of the endometrium during the IVF process of the invention. Thecultured PBMCs and/or the combined cultured and fresh PBMC compositionhave the capability to change the size and receptivity of the patient'sendometrial tissue when treated with the PBMC composition of theinvention. This results in an increase in the size, specifically anincrease in the thickness of the endometrium, as well as an enhancedability of the endometrium to bind the embryo upon implantation.

The in vitro fertilization method of the invention provides the use ofthe PBMCs obtained by the above culturing technique in order to improvepregnancy inception. After obtaining oocytes from a female patient, aportion of PBMCs is extracted from the blood of the patient. Thisportion of PBMCs is then cultured in a suitable culture media accordingto the method disclosed above to obtain a desirable quantity and qualityof PBMCs. After a predetermined waiting or delay period of timediscussed herein, an additional portion of blood is obtained from thepatient and a fresh portion of PBMCs are extracted from the blood.Alternatively, the lot of blood first obtained from the patient can bedivided into various portions containing PBMCs, of which one or some arecultured, and one or some are maintained fresh. The fresh portion ispreferably obtained on the last day of the predetermined waiting periodof time. Some or all of the fresh PCMBs are then combined with some orthe entire cultured portion of PBMCs to obtain a composition thatcontains both the fresh and the cultured PBMCs. The concentration ofPBMCs in a preferred composition of PBMCs of the invention is in therange of 4 to 5 million cells per every milliliter of the composition;however concentrations of up to 8 million cells/mL of the compositionare operable within the scope of the invention. The composition can bemixed or blended further or processed in any desirable method so as tocombine the PBMCs without damaging the cells. This composition of PBMCsis then introduced, typically via catheter injection, into the uterinecavity of the patient. According to the invention, from 20 to 72 hourslater, most preferably 24 hours, one or more embryos are transferred tothe uterus for implantation to effectuate pregnancy. The researchersherein have demonstrated that this technique enables the body to utilizethe PBMCs as building blocks to increase the thickness of the layer ofthe endometrium within the uterus and thereby to decrease the risk offailure of pregnancy inception.

It is important that the PBMCs are extracted from the patient herself inthe course of the IVF treatment of the invention. However, it isenvisioned within the scope of the invention, that the cultured portionof PBMCs can be derived from an individual other than the patientundergoing the in vitro embryo transfer. In such case, the fresh portionof PBMCs is preferably obtained from the patient undergoing embryotransfer so as to address the autoimmune response of the patient.

Preimplantation diagnostics both of embryos and of the patient'sreproductive system is another aspect of the present invention. Upondiagnosis of the patient, the thickness of the endometrium, which can betypically measured by ultrasound, is preferred to be in the range of 9mm to 11 mm at the time of embryo transfer.

Post-implantation diagnostics are also typically performed. Testing todetermine whether one or more embryos have implanted into theendometrium, i.e., whether the procedure has resulted in successfulpregnancy inception, is performed two weeks after transfer using bloodtests on b-hCG (human chorionic gonadotropin), for example, and othertechniques commonly known in the art. hCG is essential in the diagnosisof pregnancy and in of pregnancy-related conditions, such as ectopicpregnancy, spontaneous abortion, trisomy 21, molar pregnancy andchoriocarcinoma. The event of “biochemical pregnancy” occurs when apregnancy is diagnosed by detection of hCG in serum or urine that doesnot develop into a clinical pregnancy. U.S. Pat. No. 4,315,908 to Zer etal. sets forth a method for detecting hCG in the urine byradioimmunoassay. U.S. Pat. No. 8,163,508 to O'Connor et al. provides amethod and a kit for predicting pregnancy in a subject by hCG method bydetermining the amount of an early pregnancy associated isoform of hCGin a sample. Such methods of diagnosis and others are useful within thescope of the invention.

Clinicians strive to identify those embryos for transfer that are mostlikely to achieve a viable pregnancy. In certain embodiments, thepresent in invention provides for morphological, genetic and kinetictesting of the oocytes, blastocysts or embryos retrieved from thepatient and prepared in vitro. Methods of diagnosis include microscopicphysical analysis of the embryo to identify those embryos which appearto be developing normally by observation of the morphological featuresof the embryo and genetic testing of the embryo. “Preimplantationgenetic diagnosis” identifies whether the embryo contains genetic,structural, and/or chromosomal alterations or abnormalities by analysisof polar bodies, blastomeres, or trophectoderm. “Preimplantation geneticscreening” involves analysis of polar bodies, blastomeres, ortrophectoderm of embryos for detection of aneuploidy, mutation, and/orDNA rearrangement. At least three basic assisted hatching and biopsytechniques are known in the art for diagnosing embryos. These techniquesinclude mechanically creating an incision in the zona pellucida of theembryo using a specialized microsurgical knife or glass needle;chemically digesting a portion of the zona pellucida with acid, such asTyrode's solution; or removing the zona pellucida using a laser toassist separation of the blastocyst.

In a preferred embodiment, by visual observation of the embryo usingmicroscopy (for example, Nikon Eclipse TE 2000-S microscope), the embryowill display certain determined physical or morphological featuressimultaneously before it is implanted into the uterus. The state ofblastocyst maturity will be determined to be the range II AB-VI AAaccording to classification of Gardner et al, 1994, incorporated hereinby reference, whereby the intracellular mass and the thickness of thetrophectoderm layer are classified. Level VI AB represents the higheststate of blastocyst maturation, corresponding to a formed blastocystthat hatches from the zona pellucida.

Genetic diagnosis of the embryo can be performed by any techniques knownin the art, such as traditional bacterial artificial chromosome (BAC)array CGH and others like it. Microarrays, also referred to as microchiparrays, arrays or biochips, have become widely used for gene expressionand other genomic research and provide technical advantages to BAC. Amicroarray is generally made by printing or synthesizing nucleic acidthat is complementary to known sequences in a genome onto a surface. Theentire genome of an organism or parts thereof can be evaluated byhybridizing amplified and fluorescently labeled DNA (deoxyribonucleicacid) to the array. U.S. patent application Ser. No. 12/587,406,incorporated herein by reference in its entirety, discloses methods ofin vitro fertilization wherein preimplantation genetic diagnosis of all24 chromosomes of the IVF embryo is performed by whole genomeamplification and microarray analyses of polymorphisms. The methodinvolves biopsying the IVF embryo to remove one or more cells andextracting nucleic acid from the cells. Performing genome amplificationthen allows the gathering of genetic information of the embryo in orderto predict the genetic normalcy of the embryo based on the obtainedgenetic information. This technique also makes it possible to determinethe karyotype of the embryo. After the embryos are analyzed, they areranked for potential implantation into the uterus. In embodiments werethe embryos are genetically analyzed, one or more desirable embryos forimplantation into the uterus are selected based on genetic predictions.

Kinetic diagnosis of the embryo is another important procedure availablefor choosing the embryos most desirable, most healthy, for embryotransfer. One tool available is Primo Vision time-lapse embryomonitoring system (Vitrolife AB, Göteborg, Sweden). Prima Vision is acamera and computer system that is designed to capture images ofembryos, used in in vitro fertilization cycles, as they grow in theincubator. The images, taken by the camera as an embryo develops in aculture dish, are displayed on a computer screen in the laboratory. Thecomputer system not only provides the images of embryos, but alsoinformation about their pattern of growth. This information allowsembryologists and clinicians to observe healthy development in embryosand to detect any problems in the timing of cell division that mightoccur during the early stages of growth. The added benefit of PrimoVision is that it allows these observations to be made without removingthe embryos from their incubator. This allows the embryos to remain in acontrolled environment where they tend to grow faster. According to theinvention, the most desirable embryos to be selected for embryo transfershould display the following kinetics criteria: a time factor of 5-12hours for division from the 2-cell stage to the 3-cell stage; shouldhave 3 cells at 35-40 hours after zygote stage; 5 cells at 48-56 hoursafter zygote stage; 8 cells to the beginning of the third day ofdevelopment. All the cells should preferably have the same size, or asclose as possible, and the fragmentation level should be not more than5-10%.

According to the present invention, the oocytes derived from the patientor the fertilized oocytes having developed to transfer-ready embryostage are preserved during the period of time until the patient's immunesystem is sufficiently prepared to accept the embryo for gestation, forthe requisite period of time needed to suppress the autoimmune responseof the patient. Most patients desire to conceive with their own progeny,if possible, and therefore wish to utilize their own oocytes or embryos.Therefore, in most common situations, the oocytes or embryos of thepatient need to be preserved.

In a certain segment of women presenting with infertility, the woman isunable to produce eggs or to ovulate, or experiences anovulation oroligo-ovulation, so donor oocyte(s) or donor embryo(s), which areobtained from another female individual, are provided for the in vitroprocedure. It is therefore contemplated within the scope of certainembodiments of the invention that the patient will be incepted withembryos that have not been derived from the patient herself, but arederived from a different individual, therefore, a donor oocyte or donorembryo is transferred to the patient for inception.

Also within the scope of the invention are embodiments where eggs orembryo from one species is transferred to a species different than thatfrom which the egg or embryo are derived. For example, an embryo from adonkey may be implanted into the womb of a horse.

However, it will still remain the most common case that preservation ofthe oocytes or embryos, (usually derived those of the patient, butpossibly of another source) will be required. Preservation can beprovided by subjecting the oocyte or embryo to low-temperatureconditions, such as slow-cooling, rapid freezing and vitrification.Unlike sperm, which has been successfully frozen and used for years,eggs contain a great deal of water, which makes freezing more difficult.When the eggs are frozen, ice crystals can form within the egg. Theseice crystals can destroy the cell's structure. To help minimize theamount of ice crystals, scientists would remove some of the water as theegg was slowly frozen. U.S. patent application Ser. No. 10/777,149describes a method comprising centrifugation of oocytes or embryos, (forexample of dogs, cats, pigs, livestock, mice, rats, and monkeys) topolarize cytoplasmic lipid outside the oocyte or embryonic cells,subjecting the oocytes or embryos to low temperature conditions in thepresence of a cryoprotectant which results in the freezing of theoocytes or embryos prior to lipid depolarization, followed bylow-temperature storage of the frozen oocytes or embryos. However, ithas heretofore been found to be impossible to remove all the water, andthus intra- and extra-cellular ice crystal formation cannot be preventedby slow-freezing. Thus, fertilization of oocytes, embryo viability andpregnancy inception for these slow-frozen eggs, once thawed, is low.

Rapid freezing has been attempted with the use of cryoprotectants. Acryoprotectant is a substance that is used to protect biological tissuefrom freezing damage that results from ice formation. Cryoprotectantsoperate by increasing the solute concentration in cells. In order to bebiologically viable, cryoprotectants must easily penetrate the cells andnot be toxic to the cells. Conventional cryoprotectants are glycols suchas ethylene glycol, propylene glycol, and glycerol;2-methyl-2,4-pentanediol (MPD); dimethyl sulfoxide (DMSO) and sucrose.Glycerol and DMSO have been used for decades by cryobiologists to reduceice formation in sperm and embryos that are cold-preserved in liquidnitrogen. It has been found that mixtures of cryoprotectants have lesstoxicity and are more effective than single-agent cryoprotectants. Amixture of formamide with DMSO, propylene glycol, and a colloid was formany years the most effective of all artificially createdcryoprotectants. Many cryoprotectants also function by forming hydrogenbonds with biological molecules as water molecules are displaced.Hydrogen bonding in aqueous solutions is important for proper proteinand DNA function. Thus, as the cryoprotectant replaces the watermolecules, the biological material retains its native physiologicalstructure and function, although they are no longer immersed in anaqueous environment

More recently, the process of vitrification, a process of freezing andsolidification without ice crystal formation, has been applied topreserving oocytes and embryos, biological tissues, and organs fortransplant and cryonics. Some cryoprotectants function by lowering theglass transition temperature of a solution or of a material. In thisway, the cryoprotectant prevents actual freezing, and the solutionmaintains some flexibility in a glassy phase. Although slow and rapidfreezing are operable, vitrification is the preferred method ofpreservation of the oocytes or embryos pursuant to the method of theinvention. During vitrification, the oocyte or embryo is frozen quicklyenough that ice crystals do not have time to form. Any knownvitrification technique can be utilized within the method of theinvention for preservation and storage. Commonly, the oocyte or embryois first placed in a bath with a lower concentration anti-freeze, alongwith sucrose to draw water out of the oocyte or embryo. The oocyte isthen placed in a high-concentrated bath of anti-freeze for less than oneminute, while being instantaneously frozen. One example of thevitrification process operable within the process of the inventioninvolves placing embryos maintained in MEDICULT® medium at 37° C., thenplacing the embryos into the medium at 22-24° C., which is then placedin a special cryotop or cryoleaf carrier into liquid nitrogen.

According to the IVF method of the invention, the female patient isready to continue with embryo transfer when it is determined that thepatient's autoimmune system has regained hormonal balance. At such time,the cryopreservation of the eggs or the IVF-derived embryos isterminated. The oocyte(s) or embryo(s) are removed from the antifreezesolution and are thawed. Once thawed, an unfertilized egg can befertilized by any of the techniques described above by an assistedreproductive technology that injects a sperm directly into an egg. Theembryo is thawed by withdrawing it from the liquid nitrogen solution,placing into a solution having a temperature of approximately 37° C.,washing at room temperature, placing again into a solution of 37° C.,placing into a culture medium, then placing into an incubator held at atemperature similar to the internal temperature of a female uterinecavity, from 36.8 to 37.2° C. for a period of from 5 to 24 hours, mostpreferably at a temperature of 37.1° C. for 5 to 7 hours.

In the final stage of the process of the invention is the procedure bywhich one or more embryos are introduced into the patient in an attemptto produce pregnancy. This procedure is termed “embryo transfer” andinvolves transfer of the embryo into the uterus, the uterine cavity orthe fallopian tubes. Embryo transfer typically involves placing theselected embryos through the cervix into the uterine cavity of thefemale patient using a small, soft catheter and guided by an ultrasoundprobe. As provided above, according to the method of the invention,preferably at least 24 hours prior to embryo transfer, the endometriumof the uterus of the patient is prepared by injecting a composition ofPBMCs into the uterine cavity using an appropriate catheter fordelivery. The embryo is typically maintained within an embryo transfermedium, for example, MEDICULT® UTM medium, and can contain HAS,recombinant human insulin, gentamicin.

It is yet an additional aspect of the present invention that pregnancyinception is further promoted by introducing histocompatibility antigenssuch as soluble human leukocyte antigen G (sHLA-G) into the system whichfunction as “friend-or-foe communicators” to allow positive recognitionand acceptance of the embryo by the woman's immune system. HLA-G, theprecursor to its soluble form, sHLA-G, has been detected by researcherson preimplantation embryos and in the surrounding culture media obtainedduring IVF studies. These finding suggested that sHLA-G is involved fromthe early stages of pregnancy. Also suggested is the potential of sHLA-Gto operate as an indicator of embryo quality. [Menicucci et al. 1999].One study suggests that sHLA-G levels in preimplantation embryosupernatants can be quantified and suggest positive results withlikelihood of pregnancy by acting as a useful indicator of embryoquality which can be used in conjunction with morphological testing toin embryo selection criteria to increase probability of successfulimplantation. In one embodiment, s-HLA-G is added to the embryo transfermedium, with a preferred concentration measurement in the embryotransfer medium in the range of 0.175 to 0.350 optical density (OD),where OD means the optical density measured at a wavelength in the rangeof 400 to 450 nm, more specifically at a wavelength of 405 nm. Theprocedure for determining the concentration of sHLA through measurementof optical density is comprehensively described in number ofpublications. [See, for example, S. Martí et al., 2007]. The s-HLA-G isadded to the embryo transfer medium, and the embryo is then cultured insaid medium, for 5 to 20 minutes, and more preferably for approximately10 minutes immediately prior to transfer of the embryo into the uterus.

A related aspect of the invention provides a method of growing,repairing, engineering, restoring, or otherwise treating a target tissueof a patient by growing the target tissue in the presence of the PBMCcomposition according to the process set forth in this disclosure. Thetissue is grown by introducing the tissue in the presence of thecultured PBMCs or by culturing with the use of the combined PBMCcomposition of cultured and fresh PBMCs. Both in vivo and in vitroprocesses are contemplated. One embodiment of such method is that thetarget tissue is the endometrium of the uterus of a female patient.However, as disclosed in U.S. Pat. Nos. 7,795,018 and 8,216,838 toKuwana et al., the PBMCs are recognized as multipotent cells which arevery suitable for cell transplantation for organ regeneration includingbone, cartilage, skeletal muscle, fat, cardiac muscle, vascular,endothelial and neurons. Therefore, the process, the culture medium andthe composition of the invention comprising the combination of bothcultured and fresh PBMCs of the present invention can be expanded wellbeyond the boundaries of IVF treatment and treatment of the endometriumto applications involving the growth, repair and engineering of othertissues that are capable of being treated by PBMCs.

It is to be noted herein that the concept and practice of this inventionis most relevant to humans but is applicable to embryo implantation in awide variety of animals. This invention is not limited to human in vitrofertilization or embryo implantation using autoimmune system delay withuse PBMC compositions. For example, the technique can be used withanimals such as murine animals including the brown rat and the householdmouse; with companion animals including dogs and cats; and with domesticlivestock animals, such as pigs, horses, donkeys, goats, sheep, llamasand alpacas, among others in order to increase live birth rates of suchanimals. Such increases will have significant financial implications inthe livestock industry and social implications in the areas ofscientific and medical research and development.

While the invention is generally defined as above, persons skilled inthe art will appreciate that it is not limited thereto and includesembodiments of which the following examples provide further description.It is to be understood that other specific functional modifications maybe made within the scope of the art without departing from the scope ofthe present invention. The advantages of the invention are demonstratedby the examples below, which are set forth to illustrate the methods andcomposition of the invention and are intended to be purely exemplary ofthe principles and applications of the invention and are not to beviewed as limiting in its scope.

EXAMPLES

The examples below illustrate the present invention. A study wasperformed to examine and compare two IVF procedures. The first procedurewas termed the “Fresh-IVF” method and the second procedure was termedthe “Cryo-IVF” method. Statistical comparison of the clinical resultswas performed in order to examine the effect in pregnancy rates on womenpresenting with infertility of delaying embryo or blastocystimplantation into the uterine cavity of the woman after oocyteretrieval. The terms “pregnancy rate”, “clinical pregnancy rate” and“implantation rate” are used interchangeably herein and refer to thenumber of clinical pregnancies expressed per 100 embryo transfer cycles.Further investigated was the effect of intrauterine peripheralmononuclear blood cell (PMBC) administration on pregnancy rates for both“Fresh-IVF” and “Cryo-IVF” procedures.

Subjects: One-hundred and eighty (180) women presenting with infertilityin total were examined. Prior to the present study, each of the patientshad previously undergone two or more unsuccessful “Fresh-IVF” treatmentsand at least one failed “Cryo-IVF” treatment before participating in thestudy herein. The women were divided into 2 groups of 90 women pergroup. The average age of the women in the Group 1 was 35.5±3.4 years.The average age of the patients in the Group 2 was 36.5±5.5 years. Thesubjects were prepared as follows:

IVF Procedure: Standard IVF protocol using a-GnRH(gonadotropin-releasing hormone) for controlled ovary stimulation wasapplied to patients in both groups. The period of ovary stimulation forevery patient was between 10 to 12 days. The average size of follicleswas measured to be approximately 18 mm at the moment of transvaginalpuncture. Gonal, Menopur, Choragon were applied to patients of bothgroups for maintaining the luteal phase. In general, 10 to 12 eggs wereretrieved from each patient. Not less than 80% of obtained oocytes weremature enough to be fertilized (stage of maturation MII as determined byGardner's classification).

Following oocytes retrieval, the oocytes were cultured in Universal IVFMedium (MEDICULT® available from Origio A/S Corporation, Denmark) havingCO2 concentration in the range of 5.5-5.7% at a temperature of36.8-37.1° C. The oocytes were then fertilized via both ICSI andstandard IVF procedure. The technique was chosen taking into accountsperm indices, the age of the patient, the experience of previous IVFattempt with negative result. In cases of standard IVF procedure,spermatozoa were added to oocytes in UniIVF medium. The zygotesdeveloped the next day were placed into ISM-1 medium. In cases whereoocytes were obtained by transvaginal puncture, the oocytes were placedinto UniIVF medium. Following fertilization by ICSI procedure, the eggswere placed into ISM-1 immediately. The embryos were cultured inMEDICULT® Universal IVF Medium during the first three days for cleavageembryo development and in MEDICULT® BlastAssist Medium during the fourthand fifth day of culture until blastocyst formation. Embryo transfer wasperformed using MEDICULT® UTM Medium.

Vitrification: Vitrification was applied to the blastocysts or embryoson day 5 of development utilizing the standard MEDICULT® method. Theembryos were placed in specified solutions to remove as much of thewater as possible. The embryos were placed on cryotop carriers intoliquid nitrogen. Not more than two embryos were placed onto one carrier.The frozen embryos were stored during the waiting a period of three (3)months to one year.

PBMC Preparation: Peripheral blood mononuclear cells (PBMCs) wereextracted from each patient. The first portion of the PBMCs was takenfrom the patient at the day of oocytes receive in the “Fresh-IVF”protocols or three days earlier then the embryos were transferred to theuterus in the “Cryo-IVF” protocols. Initially, 10 mL of peripheral bloodwere taken from each patient. The entire quantity (10 mL) of theperipheral blood was mixed with 10 mL of MEDICULT® RPMI 1640 medium withL-glutamine and sodium bicarbonate forming a combined volume of 20 mL ofdiluted blood. Lymphocyte separation medium (density gradient medium)was then used to separate the PBMCs from the blood. A volume of 7 mL ofdiluted blood was layered onto 3 mL of the density gradient medium.After layering, centrifugation of the layered blood was performed at aspeed of 1500 rounds per minute for 30-35 min to yield the PBMCs. ThePBMCs thus obtained were washed twice in RPMI by centrifugation for 10min at a speed of 1600 rounds per minute at 4° C. The washed PBMCs weretransferred to the culture medium. The culture medium constituted amixture of RPMI 1640 medium with L-glutamine and sodium bicarbonate withaddition of human chorionic gonadotropin (hCG) and human recombinantalbumin (from Sigma-Aldrich Co., LLC). The conditions of culture weremaintained at 5.0% CO2 and 37° C. The period of PBMCs culture was 48-52hours. After 48-52 hours, an additional portion of whole blood was takenfrom the same patient. Additional PBMCs were extracted by the samemethod. The second batch of the PBMCs were combined with the culturedPBMCs and the mixture was transferred to the uterine cavity of thepatient via catheter. The procedure is carried out quickly, in a totalof 10-15 minutes. The volume of the total PCMB mixture for intrauterineapplication for each patient was in the range 0.2-0.3 mL.

Embryo Thawing and Transfer: For each patient, two blastocysts wereselected for embryo transfer. Thawing was performed using standardMEDICULT® protocol. The embryos were removed from the liquid nitrogenand placed into a solution of 37° C., then washed with several solutionsat room temperature, the placed again into a solution at 37° C., thenplaced into culture medium into an incubator at a temperature of atemperature of 37.1° C. for 6 hours. The embryos were cultured inBlastAssist MEDICULT® Medium or 3-4 hours after thawing. The embryoswere then placed into MEDICULT® UTM medium for approximately 15 minutes.Embryo transfer was performed with use of MEDICULT® UTM medium via COOK®Medical ultrasound control catheters (COOK® Medical Inc., Bloomington,Ind., U.S.).

IVF Procedure: Both groups of women underwent IVF treatment. The IVFprocedure performed on Group 1 did not involve the use of PCMBs; whereasthe IVF procedure for Group 2 was carried out with the use of PBMCs. Thecourse of treatment for each group of women included two stages—a“Fresh-IVF” cycle and a “Cryo-IVF” cycle, as described herein. On theday of embryo transfer, the endometrium size was measured at 9-11 mm inboth groups of patients. After embryo transfer, all patients wereadministered progesterone as is standard during IVF treatment in orderto prepare the endometrium for embryo implantation. Implantation of theembryo was confirmed by blood tests on b-hCG performed two weeks afterembryo transfer. Clinical pregnancy was confirmed by ultrasoundexamination three weeks after embryo transfer.

Both groups of women underwent one “Fresh-IVF” cycle with the transferof two blastocysts. Each patient was tested for pregnancy. Failureresulted when the testing showed an absence of clinical or biochemicalpregnancy. In those cases where the “Fresh-IVF” cycle failed to resultin a successful implantation of the embryo, a “Cryo-IVF” cycle wasperformed in which frozen embryos were introduced into the uterinecavity of the patient after a time-delay period of between 2-3 menstrualcycles after the negative “Fresh-IVF” cycle, which measured at aboutthree (3) months after the day on which the oocytes were retrieved fromthe ovaries of the patient.

The women in Group 1 did not receive any application of PCMBs; the womenin Group 2 received a treatment of PBMCs immediately after the end ofthe waiting period. During the “Fresh-IVF” cycles, the PBMCs wereadministered into the uterus of each subject on the second day of embryoculture at 48-52 hours after egg retrieval. During the “Cryo-IVF”cycles, the PBMCs were administered to uterus about 24 hours beforeembryo transfer.

Results: The group of patients undergoing Fresh-IVF treatment withoutPBMCs, resulted in an implantation rate of 22.2% (20 clinicalpregnancies after 90 ET). The use of PMBCs during the “Fresh-IVF” methodcaused an increase in the implantation rate up to 31.1% (28 clinicalpregnancies after 90 ET). In the group of women undergoing attempts via“Cryo-IVF” treatment (time delay lasting at least 3 months for bothGroup 1 and Group 2), the implantation rate without intrauterineapplication of PBMCs was 21.4% (15 clinical pregnancies after 70 ET),while after application of PBMCs, the implantation rate was almost twotimes higher 41.9% (26 clinical pregnancies after 62 ET). The totalsuccess rate for Group 1 (90 patients without application of PBMCs) was38.9% (35 clinical pregnancies). The total success rate for Group 2 (90patients with application of PBMCs) was 60.0% (54 clinical pregnancies).The clinical results of the study are presented in Table 1 below. Theabbreviation “ET” indicates “embryo transfers”.

TABLE 1 Implantation rates in “Fresh-IVF” and “Cryo-IVF” cycles, withand without administration of PBMCs to the patient. Clinical PregnancyRate Group 1 (without PBMCs) Group 2 (with PBMCs) Fresh-IVF 22.2% (20pregnancies after 31.1% (28 pregnancies after 90 ET) 90 ET) Cryo-IVF21.4% (15 pregnancies after 41.9% (26 pregnancies after 70 ET) 62 ET)Total 38.9% (35 pregnancies for 90 60.0% (54 pregnancies for 90patients) patients)

Table 1 demonstrates the increased pregnancy rate obtained when comparedto traditional IVF through the use of the woman's own PBMCs through thepreparation of uterus prior to embryo transfer. It is furtherdemonstrated that combining the delayed or “Cryo-IVF” technique togetherwith preparation for inception of a patient's uterus by theadministration of the woman's own PBMCs during the in vitrofertilization process produces a significant increase in the rate ofpregnancy of infertile women as compared to previously-known IVFprocedures, possibly as a synergistic result both of allowing the femalepatient's autoimmune system to regain hormonal balance and allowing theinception process to be realized in a uterine cavity having an enhancedability for embryo implantation.

Any reference in this specification to “one embodiment,” “anembodiment,” “example embodiment,” etc., means that a particularfeature, structure, or characteristic described in connection with theembodiment is included in at least one embodiment of the invention. Theappearances of such phrases in various places in the specification arenot necessarily all referring to the same embodiment. Further, when aparticular feature, structure, or characteristic is described inconnection with any embodiment, it is submitted that it is within thepurview of one skilled in the art to effect such feature, structure, orcharacteristic in connection with other ones of the embodiments.

Although embodiments have been described with reference to a number ofillustrative embodiments thereof, it should be understood that numerousother modifications and embodiments can be devised by those skilled inthe art that will fall within the spirit and scope of the principles ofthis disclosure. More particularly, various variations and modificationsare possible in the component parts and/or arrangements of the subjectcombination arrangement within the scope of the disclosure, the drawingsand the appended claims. In addition to variations and modifications inthe component parts and/or arrangements, alternative uses will also beapparent to those skilled in the art.

What is claimed is:
 1. A method of in vitro fertilization for a femalepatient, the method comprising: (a) introducing into the uterus aneffective amount of a composition comprising peripheral bloodmononuclear cells, and (b) transferring at least one embryo into theuterus of the patient, wherein the at least one embryo is derived fromat least one oocyte retrieved from the patient at least two months priorto the transferring of the at least one embryo, wherein: prior tointroducing the composition comprising peripheral blood mononuclearcells into the uterus, at least a portion of the peripheral bloodmononuclear cells were cultured in the presence of human chorionicgonadotropin; the peripheral blood mononuclear cells are derived fromblood collected from the patient at a first time between (i)transferring the at least one embryo into the uterus of the patient and(ii) three days prior to transferring the at least one embryo into theuterus of the patient; and the method results in an increase in theprobability of implantation of the at least one embryo in the uteruswith successful inception of pregnancy when compared to in vitrofertilization methods lacking at least one of (a) and (b).
 2. The methodof claim 1, wherein the at least two month period of time between theretrieval of the at least one oocyte from the patient and thetransferring of the at least one embryo is an amount of time sufficientto decrease the risk of autoimmune response of the patient.
 3. Themethod of claim 1, wherein the at least two month period of time betweenthe retrieval of the at least one oocyte from the patient and thetransferring of the at least one embryo corresponds to at least twomenstrual cycles or two cycles of ovulation of the patient.
 4. Themethod of claim 1, wherein the at least one embryo is transferred to theuterus within an embryo transfer medium and the embryo transfer mediumcomprises an inception-promoting agent.
 5. The method of claim 4,wherein the patient is a human, the inception-promoting agent is solublehuman leukocyte antigen G (sHLA-G) and the concentration of the sHLA-Gin the embryo transfer medium is in the range 0.175-0.350 OD, where ODmeans the optical density measured at a wavelength in the range of 400to 450 nm.
 6. The method of claim 5, further comprising culturing the atleast one embryo in said embryo transfer medium for a period of time inthe range of 5 min to 20 min immediately prior to transferring the atleast one embryo into the uterus.
 7. The method of claim 1, wherein thepatient is of human, murine, or livestock animal origin.
 8. The methodof claim 1, wherein the at least one oocyte is retrieved from thepatient two to twelve months prior to the transferring of the at leastone embryo.
 9. The method of claim 8, wherein during the two to twelvemonths prior to the transferring of the at least one embryo, controlledovarian stimulation was not performed on the patient.
 10. The method ofclaim 1, wherein the at least one oocyte is retrieved from the patientthree to twelve months prior to the transferring of the at least oneembryo.
 11. The method of claim 1, wherein during the at least twomonths prior to the transferring of the at least one embryo, controlledovarian stimulation was not performed on the patient.
 12. The method ofclaim 1, wherein: a first portion of the peripheral blood mononuclearcells is derived from the blood collected from the patient at the firsttime between (i) transferring the at least one embryo into the uterus ofthe patient and (ii) three days prior to transferring the at least oneembryo into the uterus of the patient, a second portion of theperipheral blood mononuclear cells is derived from blood collected fromthe patient at a second time between (i) transferring the at least oneembryo into the uterus of the patient and (ii) three days prior totransferring the at least one embryo into the uterus of the patient, andthe second time is after the first time.
 13. The method of claim 12,wherein the first portion of the peripheral blood mononuclear cells isobtained by the culturing, in the presence of human chorionicgonadotropin, of the peripheral blood mononuclear cells from the bloodcollected from the patient at the first time.
 14. A method of in vitrofertilization for a female patient, the method comprising: (a)initiating ovulation in the patient to obtain at least one oocyte, (b)obtaining peripheral blood mononuclear cells from the patient (PBMCs),(c) fertilizing the at least one oocyte obtained in (a) with spermatozoato create at least one embryo, (d) introducing the patient's PBMCs intothe uterus of the patient at least two months after (a), and (e)introducing the at least one embryo of (c) into the uterus of the femalepatient after (d) to effectuate implantation of the at least one embryoin the uterus, wherein: at least a portion of the PBMCs obtained fromthe patient in (b) were cultured in the presence of human chorionicgonadotropin before introducing the PBMCs into the uterus of the patientin (d): and the obtaining of the PBMCs from the patient in (b) wasperformed at most three days prior to introducing the at least oneembryo into the uterus of the patient in (e).
 15. The method of claim14, wherein the at least one embryo is introduced into the uterus ofsaid patient 20-24 hours after (d).
 16. The method of claim 14, whereinin (d) the patient's PBMCs are introduced into the uterus of the patienttwo to twelve months after (a).
 17. The method of claim 16, whereinduring the two to twelve months between (a) and (d), controlled ovarianstimulation was not performed on the patient.
 18. The method of claim14, wherein in (d) the patient's PBMCs are introduced into the uterus ofthe patient three to twelve months after (a).
 19. The method of claim14, wherein during the at least two months between (a) and (d),controlled ovarian stimulation was not performed on the patient.
 20. Themethod of claim 14, wherein: a first portion of the PBMCs introducedinto the uterus of the patient in (d) is derived from blood collectedfrom the patient at a first time between (i) introducing the at leastone embryo into the uterus of the patient in (e) and (ii) three daysprior to introducing the at least one embryo into the uterus of thepatient in (e), a second portion of the PBMCs introduced into the uterusof the patient in (d) is derived from blood collected from the patientat a second time between (i) introducing the at least one embryo intothe uterus of the patient in (e) and (ii) three days prior tointroducing the at least one embryo into the uterus of the patient in(e), and the second time is after the first time.
 21. The method ofclaim 20, wherein the first portion of the PBMCs is obtained by theculturing, in the presence of human chorionic gonadotropin, of the PBMCsfrom the blood collected from the patient at the first time.